Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in factor VIII. The activated form of factor VIII, factor VIIIa, functions as a cofactor for the factor IXa-dependent activation of factor X, increasing the kcat for this reaction by several orders of magnitude. On-going research in our laboratory has focused upon this clinically important and biochemically fascinating protein. We propose to elucidate fine point structural details of inter-protein interactions that will define mechanisms for catalytic rate enhancement and the regulation of activity of the intrinsic factor Xase. Aim I will study the role of factor VIIIa (subunits) in the interaction with factor IXa and modulation of its active site. Studies will focus on the A2 subunit based upon our observations that this subunit contains an extended factor IXa-interactive surface and that isolated A2 enhances the kcat for factor IXa-catalyzed activation of factor X. A primary effort will focus on experimentally assessing and refining our model for the A2-factor IXa interface. Proteolytic conversion of procofactor to active factor Villa exposes a functional factor IXa-interactive site on the A2 domain that is otherwise cryptic, and we propose to identify this critical region(s) thereby defining the molecular mechanism for factor VIII "activation." Functional assays using native and mutant proteins will be complemented with physical methods such as fluorescence-based assays, chemical crosslinking, hydrogen/deuterium exchange and mass spectrometry to assess important residues/regions that participate in the extended interactive surface. Related studies will determine the bases for the factor VIIIa-dependent contributions to the Km of factor Xase for factor X and for a novel substrate electrostatic steering mechanism. Aim II will examine regulation of factor VIIIa by the inactivating proteinases activated protein C (APC) and factor Xa, with emphasis on examining interactions of exosites using unique proteinase forms. Mechanisms for these interactions leading to factor VIIIa inactivation, and hence factor Xase regulation remain poorly understood. We will probe these interactions using native and recombinant factor VIIIa (and isolated subunits) possessing alterations in putative exosite-interactive regions as well as cleavage-resistant forms. A focal point is the study of proteolysis at Arg336 in the A1 subunit, a predominant site cleaved by both APC and factor Xa, and the influence of attack at this site on cleavage at more secondary sites. Definition of these issues will yield valuable and fundamental insights into the biochemistry of the native as well as dysfunctional factor VIII molecules, and provide information for the design of superior therapeutics